Survival rates drop precipitously when a tumor acquires the ability to metastasize to distant organs. The complexity of the metastatic process has, to date, precluded the approval therapeutics that specifically target metastasis. New options for treatment will become available as our mechanistic understanding of metastasis improves. This application responds to the Provocative Question #24: Given the difficulty of studying metastasis, can we develop new approaches, such as engineered tissue grafts, to investigate the biology of tumor spread? The vascular supply to a tumor is a major route for metastasis as motile cancer cells that have undergone epithelial mesenchymal transition (EMT) can intravasate into the vessels and be transported through the circulation to a distant site, such as the liver. The vascular supply to the tumor is extremely leaky and comprised of tortuous and unorganized vessels that have a profound impact on the tumor microenvironment including the concentration of growth factors (e.g., transforming growth factor , TGF), interstitial pressure, and oxygen content. There are currently no in vitro models that recapitulate these prominent features of the tumor microenvironment, and thus the early events of metastasis. Employing microfluidic and tissue engineering technologies, our team of investigators has recently developed an in vitro high-throughput model of human microtissues (~ 1 mm3) that is perfused by living dynamic human microvessels. The model is primed to incorporate tumor spheroids and create a novel in vitro model of the tumor microenvironment that includes the essential feature of flow through human microvessels. The central objective of our application is to create a high-throughput platform of 3-D human tumor spheroids perfused by human microvessels that mimics EMT and intravasation. We hypothesize that intraluminal fluid shear stress impacts the efficiency of intravasation and tumor cell exit into the systemic circulation. This hypothesis can be uniquely addressed by our in vitro platform. The specific aims focus on platform development, validation, and investigation: 1) develop and optimize a 3-D model of perfused human tumors by incorporating epithelial- derived tumor spheroids (human colorectal cancer) into a perfused network of 3-D human microtissues; 2) validate an in vitro platform of tumor metastasis by stimulating (with TGF, and/or hypoxia) and characterizing EMT and intravasation of human tumor spheroids within 3-D perfused microtissues; and 3) test the hypothesis that intraluminal fluid shear impacts endothelial permeability by affecting intercellula junctional integrity, and thus the efficiency of intravasation and exit into the systemic circulation. Completing the specific aims will create and validate a new in vitro model of tumor metastasis that will significantly enhance the temporal and spatial resolution of the process. Finally, the model is flexible and, although our application will focus on intravasation, the model could also be used to examine other steps in metastasis such as extravasation and survival at a distant site.